The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Homogeneous Bioassay Technologies
Development of ever more simple, rapid, reliable and sensitive, quantitative bioassays has been one of the main objects in evolution of immunoassay, other ligand binding assay and biological activity assay techniques. Homogeneous immunoassay methods have received much attention, because they could speed up an assay by eliminating the need for cumbersome steps of separation of bound and free label, and significantly simplify construction of an instrument required to perform an assay automatically (Ullman E F, J Chem Ed 1999; 76:781-788; Ullman, E F, J Clin Ligand Assay 1999; 22:221-227). One of the major obstacles in the development of advanced, low-cost point-of-care instruments for immunoanalysis are the limitations of label technologies. Currently available label technologies suitable for homogeneous, non-separation immunoassays either suffer from interference of complex biological sample matrices, the technologies cannot be universally employed to different analytes and assay formats, e.g. both competitive and non-competitive assays, they simply do not enable sensitive assays enough to be performed using a rapid read-out, or the instrumentation required for detection is too complex or expensive to be employed in a point-of-care instrument.
Homogeneous assay methods based on photoluminescence have received much attention, since several types of physical and chemical interactions can be employed to modulate the emission of photoluminescent labels due to formation of specific immunological complexes. The commonly employed methods are based on polarization of the emitted light or nonradiative energy-transfer between two photoluminescent compounds or between a photoluminescent and a non-luminescent compound (Hemmilä I, Clin Chem 1985; 31:359-370). Fluorescence properties of two fluorescent compounds were employed in a homogeneous immunoassay in late 1970's when Ullman et al. demonstrated, that fluorescence energy transfer between fluorescein donor and tetramethylrhodamine acceptor pair could be employed to construct both competitive and non-competitive immunoassays (Ullman E F et al. J Biol Chem 1976; 251:4172-4178; Ullman E F & Khanna P L, Methods Enzymol 1981; 74:28-60). The energy transfer was measured from decrease in the fluorescence of donor, which limited further improvements in sensitivity. Increase in the fluorescence of the acceptor was not practicable, since only a little increase in a sensitised acceptor emission could be observed over autofluorescence, light scattering or absorbance of biological sample matrices and the direct emission of the donor at acceptor-specific wavelength.
Many compounds and proteins present in biological fluids or serum are naturally fluorescent, and the use of conventional fluorophores leads to serious limitations of sensitivity (Wu P and Brand L, Anal Biochem 1994; 218:1-13). Another major problem when using homogeneous fluorescence techniques based on intensity measurements is the inner filter effect and the variability of the optical properties of a sample. Sample dilution has been used to correct this drawback, but always at the expense of analytical sensitivity. Feasibility of fluorescence energy transfer in immunoassays was significantly improved when fluorescent lanthanide cryptates and chelates with long-lifetime emission and large Stokes' shift were employed as donors in the 1990's (Mathis G, Clin Chem 1993; 39:1953-1959; Selvin P R et al., Proc Natl Acad Sci U S A 1994; 91:10024-10028; Stenroos K et al., Cytokine 1998; 10:495-499; WO 98/15830; U.S. Pat. No. 5,998,146; WO 87/07955). Feasibility of the label technology in dissociation reactions, e.g. cleavage assays, has also been described (Karvinen J et al., J Biomol Screen 2002; 7:223-231).
Time-resolved fluorescence detection of sensitized emission allowed elimination of the autofluorescence and dual signal ratio measurement (U.S. Pat. No. 5,527,684; Mathis, G, Clin Chem 1993; 39:1953-1959) corrected the variability of optical properties of the sample. Fluorescence of the compounds and proteins present in biological fluids has a short lifetime and the use of long-lifetime labels combined with time-resolved detection of the sensitised (prolonged lifetime) acceptor emission allowed minimization of the assay background and improved signal to background ratio. The variability of absorption of excitation light at 337 nm was corrected by measuring the emission of the donor at 620 nm and using the ratio of the energy transfer signal at 665 nm and the emission at 620 nm to generate a variable that is independent of the optical properties of the serum sample.
Separation free assay technologies based on confocal detection of photoluminescent labels bound on particulate carriers have been introduced as an alternative to real homogeneous assays (Saunders G C et al., Clin Chem 1985; 31:2020-2023; Frengen J et al., Clin Chem 1993; 39:2174-2181; Fulton R J et al., Clin Chem 1997; 43:1749-1756). In recent years, the technology has been developed, and some novel carrier-based immunoassays can be considered as homogeneous assays, since they are practically similar to perform (Hanninen P et al., Nat Biotechnol 2000; 18:548; U.S. Pat. No. 5,891,738; Schaertl S et al., J Biomol Screen 2000; 5:227-238), although the actual signal of the label is not modulated, but the unbound labelled component is spatially excluded from measurement. These assay are otherwise comparable to homogeneous assays, but measurement is relatively slow, since carrier particles have to be either actively scanned or passively diffuse to focal point, and signal associated to several carrier particles is required for reliable measurement (Waris M E et al., Anal Biochem 2002; 309:67-74). Luminescence oxygen channelling immunoassay, LOCI, a true homogeneous assay based on particulate label pair and photoactivated chemiluminescence with up-conversion has been demonstrated with extreme sensitivity (Ullman E F et al., Clin Chem 1996; 42:1518-1526; Ullman E F et al., Proc Natl Acad Sci USA 1994; 91:5426-5430), but the reservations in the particle-particle pair formation and susceptibility to sample interferences have prevented the adaptation of the technology for routine diagnostic applications. To avoid sterical hindrance in binding at least one of the labels, preferably both labels of a label-pair should be of small molecular size.
The homogeneous assays techniques based on fluorescence labels would enable very rapid and simple assays using a single incubation method without any wash steps. In most of the conventional homogeneous fluorescence immunoassay technologies, the assay performance has still severe limitations: the sensitivity of the assays is limited by interferences from matrix components and optical properties of matrices, e.g. urine, saliva, serum, plasma or whole blood, to fluorescence yield and level of background, and by the attainable degree of fluorescence modulation, e.g. quenching, enhancement, energy transfer or polarization (Hemmilä I, 1985). Optical properties of biological matrices at visible wavelengths and in the near-infrared region are described in Chance B, Photon Migration in Tissues, pp. 206; Kluwer Academic/Plenum Publishers, 1990, New York. Fluorescence polarization assays utilizing fluorescence utilizing near-infrared fluorophores (U.S. Pat. No. 6,060,598) are limited to competitive binding assays. Ideally, the modulation of fluorescence signal should not restrict the type of assay, non-competitive or competitive, or type and molecular size of an analyte, and the modulation should stay detectable and preferable unchanged when a significant portion of the assay solution consists of biological matrix. These stringent requirements for a general and sensitive homogeneous fluorescence immunoassay explain, why the majority of developed assays have been limited to academic research, restricted to particular type of analyte and matrix, and have weak sensitivity and narrow dynamic range (Mathis G, 1993).
Long Lifetime Luminescent Labels
Employment of long-lifetime fluorescent lanthanide cryptates in homogeneous energy transfer immunoassay provided an improved assay technology, which solved most of the problems associated with the earlier homogeneous immunoassay methods. Time-resolved amplified cryptate emission (TRACE) technology is a general label technique enabling highly sensitive non-competitive assays and it is also suitable for competitive assays. The technology is applicable to serum samples only with correction of sample absorbance using simultaneous measurement of both the donor and the acceptor emission and it is not applicable to whole-blood samples. Although this technology has enabled rapid and simple immunoassay from serum with good precision, it has not been a commercial success. The instrument employed nitrogen laser to enable an extremely powerful and sharp excitation pulse and immediate opening the measurement window after an excitation pulse and resulted in excellent performance but expensive design of instrument. Further, the advantages of the rapid assay were partly diminished, as the technology was not suitable for whole blood and required preparation of serum sample.
Development of homogeneous whole blood analysis using photoluminescent proximity-based techniques requires selection of labels with excitation and sensitised emission at near-infrared window. In principle, suitable short-lifetime near-infrared fluorescent donor and acceptor dyes exist, but the autofluorescence and scattering still restrict the limit of detection (Oswald B et al., Anal Biochem 2000; 280:272-277), although pulsed laser or high-frequency modulated excitation and sub-nanosecond time-resolution in fluorescence detection (Augustin C. 2001, Ruthenium-ligand complexes as bioanalytical luminescent probes for polarization and energy transfer systems. Ph.D. Thesis, University of Regensburg, Germany, 119 pp.), available at high cost, would somewhat improve the sensitivity. Inexpensive instrumentation would be available for millisecond time-resolution, but unfortunately no perfect near-infrared fluorescent donor dyes are available with millisecond fluorescence lifetime. Neither near-infrared fluorescent lanthanide(III) chelates (Werts M H V. 2000. Luminescent lanthanide complexes: Visible light sensitised red and near-infrared luminescence. Ph.D. Thesis, University of Amsterdam, The Netherlands. 131 pp.), ruthenium(II) complexes (Augustin C M et al., Anal Biochem 2002; 305:166-172), phosphorescent platinum(II) and palladium(II) porphyrins (Soini A E et al., J Porphyrins Phthalocyanines 2001; 5:735-741) nor energy-transfer dyes (Lakowicz J R et al. Anal Biochem 2001; 288:62-75) provide all the required features, since they are excited either outside the near-infrared window or the emission peaks are at wavelengths over 850 nm, where no feasible acceptor dyes are currently available.
Anti-Stokes or Up-Converting Phosphors
Luminescent materials which can be excited by long-wave radiation, for example, infra-red radiation and then emit radiation having shorter wavelengths, particularly visible radiation are also called anti-stokes phosphors or up-converting phosphors. They are excited by sequential absorption of two or more photons of the exciting radiation. The excitation is thus effected in two or more stages so that the luminescent centres of the phosphors reach such a high energy level that photons emitted from the same are richer in energy than the exciting photons, i.e. the emitted radiation has a shorter wavelength than the exciting radiation. The two or more absorbed photons may have the same or different energy or wavelength and they may be produced by a single or multiple light sources.
The up-conversion differs from two-photon or multi-photon excitation based on simultaneous multi-photon absorption (U.S. Pat. No. 5,777,732, U.S. Pat. No. 5,523,573) as the absorption of multiple photons in the described method does not need to be simultaneous and significantly lower intensities of excitation light can be applied. Excitation of up-converting labels can be performed with e.g. pulsed halogen lamps or semiconductor light-emitting diodes or lasers, which are compact, have high power and are also inexpensive (Johnson B D, Photonics Spectra 2001; 35:52). The exciting radiation employed in the up-conversion is not sufficiently energetic to excite background emission from the sample or surroundings with multi-photon excitation at a wavelength, which would interfere with the measurement.
The up-converting phosphors has been known since 1970's, but their unique property of anti-stokes fluorescence has not been employed in biomedical research until 1990's (Corstjens P et al., Clin Chem 2001; 47:1885-1893; Niedbala R S et al., Anal Biochem 2001; 293:22-30; van De Rijke F et al., Nat Biotechnol 2001; 19:273-276; Zijlmans HJMAA et al., Anal Biochem 1999; 267:30-36).
Employment of long-lifetime particulate donor with short lifetime fluorescent acceptor in ligand binding assays is described in WO 02/44725, which also covers the use of anti-stokes phosphors as donors, but it does not relate to whole-blood analysis. The description of particulate-based homogeneous time-resolved assay in this patent application is partly overlapping with earlier patent applications, WO 00/23785 and WO 99/12018, which, however, do not consider the use of anti-stokes phosphors. The use of up-converting phosphors in separation based diagnostic applications has been described in WO 94/07142, U.S. Pat. No. 5,674,698, U.S. Pat. No. 6,159,686 and U.S. Pat. No. 6,312,914. The homogeneous assay principle based on up-converting phosphors has been described in WO 98/43072 and in more detail in US 2002/0119485. Up-converting chelates have been described in U.S. Pat. No. 5,891,656 and Faris G W and Hryndza M, Proc SPIE—Int Soc Opt Eng 2002; 4626:449-452. Method of separation of different lifetimes using cyclical excitation has been described in WO 99/63327.
Thus, these up-converting phosphors and up-converting chelates have been suggested for use in various assays, also homogeneous energy transfer assays, but so far they have not been suggested for use in homogeneous energy transfer assays to be carried out in whole blood, serum or plasma or other biological fluids, which absorb radiation in the wavelength range 300 to 600 nm and are difficult to be measured with current homogeneous bioassay technologies.